Optimization of Thin-layer Chromatography and High-Performance Liquid Chromatographic Method for Piper guineense Extracts

Abstract

In this study, a thin-layer chromatography (TLC) and a high performance liquid chromatographic (HPLC) methods were developed for the chemical profiling, qualitative and quantitative analysis of P. guineense extracts. To obtain a chromatogram with satisfactory resolution and favorable retention time, DryLab software was used to simulate and optimize a HPLC method for the analysis of P. guineense extracts. The aim was to achieve the best possible overall resolution while keeping the analysis time and solvent consumption to a minimum. With the optimized method, a total of 16 main components in the extract were separated with favorable resolution. Optimal TLC conditions were also developed using solvents of various solvent strength (ST) and solvent selectivity (PS) values. The mobile phase composition was systematically tested using various proportions of solvents differing in ST and PS values under the same experimental conditions. During the optimization, emphasis was set on achieving the best possible overall separation of the main components of the extracts (for example piperine). In addition, the effects of the developing chamber was tested using three types of unsaturated chamber conditions: horizontal chamber in sandwich configuration, horizontal chamber in non-sandwich configuration and twin-trough vertical chamber. During the study, a TLC method was developed, and the best mobile-phase composition giving favorable resolution of the bands was toluene: ethyl acetate (PS 6-4 corresponding to 60:40 % v/v). The developing chamber conditions did not affect the TLC separation efficacy in the analysis of P. guineense extracts. The HPLC method was applied to determine the percentage content of piperine in P. guineense. The piperine content was 0.43 % w/w, linearity (0.997), interday precision (% relative standard deviation (RSD), 1.6), intraday precision (% RSD, 2.7 – 5.9), recovery (98.4%), limit of detection (0.001 μg /mL) and limit of quantification (0.003 μg /mL).